缺氧缺血性脑损伤（HIBD）是新生儿远期行为异常的主要原因，小胶质细胞（MC）活化及炎症反应为其关键。CD11b系MC标记物，前期研究发现HIBD后MC活跃、外周血单核细胞（PBMCs）入侵脑内并表达CD11b但功能不清。MC对突触的修剪系脑发育所必需，但其在HIBD中的变化不明。CD200R能调控脑内炎症反应但对突触修剪的影响有待研究。项目拟揭示CD200R对HIBD后MC及PBMCs突触修剪的作用。采用Cx3cr1GFP/+Ccr2RFP/+小鼠构建模型，以区分MC及PBMCs，对二者炎症反应蛋白、吞噬功能、突触修剪细胞数进行分析，揭示它们在HIBD炎症反应及突触修剪中的贡献；采用CD11b磁珠提取结合流式分选出MC及PBMCs，分别与神经元共培养观察对轴突清除和生长的作用，并采用CD200R阻断抗体阐明CD200R对MC及PBMCs突触修剪的调控，为HIBD远期行为障碍的机制提供证据

Hypoxia and ischemia brain damage (HIBD) is the major cause of long-term behavior deficit in neonates. Microglial cell (MC) activation and its related inflammatory response are believed as a key mechanism of HIBD. CD11b is a marker of MC, interestingly, our previously study found that peripheral blood monocyte cells (PBMCs) infiltrated into brain and expressed with CD11b in a rodent model of HIBD. MC-mediated synapse pruning plays fundamental contributes in normal brain developing. However, whether and how MC responses to HIBD and modulates synapses in HIBD-affected subjects remains uncovered. CD200R plays key roles in modulate brain inflammation but its role in synapses pruning is unclear. In this proposal, we focus on CD200R’s modulatory mechanism in MC- and PBMCs-induced synaptic pruning after HIBD. In order to distinguish MC and PBMCs, we will apply Cx3cr1GFP/+Ccr2RFP/+ double transgenic mice to develop HIBD model. In an in vivo study, inflammatory protein expression, synapse number, and phagocytic ability of MC or PBMCs will be measured after HIBD. In the in vitro system, we will use CD11b magnetic beads to isolate CD11b+ cell from HIBD mice brain. Microfluidic will be adopted to establish MC and neuron co-culture or PBMCs and neuron co-culture systems. Within these in vitro co-cultures, MC or PBMCs synapse pruning function will be tested via axon clearance and axon growth. CD200R antibody will be used to test CD200R’s role in modulating MC- or PBMCs-mediated synaptic pruning after HIBD. The completion of this proposal will shield lights on synaptic pruning after HIBD and may help to develop potential therapeutic strategy for HIBD treatment.Inflammatory protein, synapse number and phagocytic ability of MC or PBMCs will be used to evaluate inflammation and synaptic pruning function change of MC or PBMCs after HIBD in vivo. Along with it, CD11b magnetic beads will be applied to isolate CD11b+ cell from HIBD mice brain. Microfluidic will be adopted to establish MC or PBMCs and neuron co-culture system and axon clearance and axon growth experiment will be used to test MC or PBMCs synapse pruning function in vitro. CD200R blockage antibody will be applied in order to test CD200R function in modulate MC/PBMCs synaptic pruning function in the brain after HIBD. The successful completion of this proposal will shield lights on synaptic pruning after HIBD and may help to develop potential therapeutic strategy for HIBD treatment.